Heat sensitivity of Rubisco,Rubisco activase and Rubisco binding protein in higher plants

During the past few years the investigations concerning Rubisco and the changes of its activity and properties at elevated temperature were reconsidered with special reference to the important role of Rubisco activase and Rubisco binding protein. The major changes in Rubisco, Rubisco activase and Rubisco binding protein reported recently are presented in this review. New information on these proteins, including their changes under heat stress conditions, is discussed together with open questions.     

Influence of ozone on transgenic tobacco plants expressing reduced quantities of Rubisco

RbcS-antisense transformed tobacco plants (Nicotiana tabacum cv. Petit Havana) expressing reduced quantities of Rubisco protein were used to examine the role of Rubisco quantity in determining ozone (O₃) sensitivity. Transformed and wild-type plants were exposed to O₃ in the greenhouse and in the field. Stomatal conductance, net photosynthesis and Rubisco protein quantity were measured at various times. Antisense-transformed genotypes responded to O₃ by exhibiting rapid, severe foliar necrosis. The wild-type plants responded more slowly, exhibiting limited injury. Decreases in stomatal conductance, net photosynthesis or Rubisco quantity in plants exposed to O₃ were not observed in asymptomatic leaves. Total biomass was lower for the transformed genotypes and decreased in both genotypes after exposure to O₃. Shoot–root ratio and specific leaf area were higher in the transformed genotypes and increased in both genotypes with exposure to O₃. Measurements of intercellular airspace demonstrated the presence of larger intercellular spaces in the transformed plants. The indirect effects of the rbcS antisense transformation, including morphological changes in the leaf, probably rendered the transformed plants more sensitive to the oxidant. The decreased quantity of Rubisco is not thought to be directly related to increased O₃ sensitivity in the transformed plants.     

Antisense inhibition of Rubisco activase increases Rubisco content and alters the proportion of Rubisco activase in stroma and thylakoids in chloroplasts of rice leaves

BACKGROUND AND AIMS: Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase (RCA) is a nuclear-encoded chloroplast protein that modifies the conformation of Rubisco, releases inhibitors from active sites, and increases enzymatic activity. It appears to have other functions, e.g. in gibberellin signalling and as a molecular chaperone, which are related to its distribution within the chloroplast. The aim of this research was to resolve uncertainty about the localization of RCA, and to determine whether the distributions of Rubisco and RCA were altered when RCA content was reduced. The monocotyledon, Oryza sativa was used as a model species. METHODS: Gas exchange and Rubisco were measured, and the sub-cellular locations of Rubisco and RCA were determined using immunogold-labelling electron microscopy, in wild-type and antisense rca rice plants. KEY RESULTS: In antisense rca plants, net photosynthetic rate and the initial Rubisco activity decreased much less than RCA content. Immunocytolocalization showed that Rubisco in wild-type and antisense plants was localized in the stroma of chloroplasts. However, the amount of Rubisco in the antisense rca plants was greater than in the wild-type plants. RCA was detected in both the chloroplast stroma and in the thylakoid membranes of wild-type plants. The percentage of RCA labelling in the thylakoid membrane was shown to be substantially decreased, while the fraction in the stroma was increased, by the antisense rca treatment. CONCLUSIONS: From the changes in RCA distribution and alterations in Rubisco activity, RCA in the stroma of the chloroplast probably contributes to the activation of Rubisco, and RCA in thylakoids compensates for the reduction of RCA in the stroma, allowing steady-state photosynthesis to be maintained when RCA is depleted. RCA may also have a second role in protecting membranes against environmental stresses as a chaperone.     

Phosphorylation pattern of Rubisco activase in Arabidopsis leaves

Rubisco activase (RCA) is an ancillary photosynthetic protein essential for Rubisco activity. Some data suggest that post‐translational modifications (such as reduction of disulphide bridges) are involved in the regulation of RCA activity. However, despite the key role of protein phosphorylation in general metabolic regulation, RCA phosphorylation has not been well characterised. We took advantage of phosphoproteomics and gas exchange analyses with instant sampling adapted to Arabidopsis rosettes to examine the occurrence and variations of phosphopeptides associated with RCA in different photosynthetic contexts (CO₂ mole fraction, light and dark). We detected two phosphopeptides from RCA corresponding to residues Thr 78 and Ser 172, and show that the former is considerably more phosphorylated in the dark than in the light, while the latter show no light/dark pattern. The CO₂ mole fraction did not influence phosphorylation of either residue. Phosphorylation thus appears to be a potential mechanism associated with RCA dark inactivation, when Rubisco‐catalysed carboxylation is arrested. Since Thr 78 and Ser 172 are located in the N and Walker domains of the protein, respectively, the involvement of phosphorylation in protein–protein interaction and catalysis is likely.     

A role for differential Rubisco activase isoform expression in C4 bioenergy grasses at high temperature

Rubisco activase (Rca) facilitates the release of sugar‐phosphate inhibitors at Rubisco catalytic sites during CO₂ fixation. Most plant species express two Rca isoforms, the larger Rca‐α and the shorter Rca‐β, either by alternative splicing from a single gene or expression from separate genes. The mechanism of Rubisco activation by Rca isoforms has been intensively studied in C₃ plants. However, the functional role of Rca in C₄ plants where Rubisco and Rca are located in a much higher [CO₂] compartment is less clear. In this study, we selected four C₄ bioenergy grasses and the model C₄ grass setaria (Setaria viridis) to investigate the role of Rca in C₄ photosynthesis. All five C₄ grass species contained two Rca genes, one encoding Rca‐α and the other Rca‐β, which were positioned closely together in the genomes. A variety of abiotic stress‐related motifs were identified in the Rca‐α promoter of each grass, and while the Rca‐β gene was constantly highly expressed at ambient temperature, Rca‐α isoforms were expressed only at high temperature but never surpassed 30% of Rca‐β content. The pattern of Rca‐α induction on transition to high temperature and reduction on return to ambient temperature was the same in all five C₄ grasses. In sorghum (Sorghum bicolor), sugarcane (Saccharum officinarum), and setaria, the induction rate of Rca‐α was similar to the recovery rate of photosynthesis and Rubisco activation at high temperature. This association between Rca‐α isoform expression and maintenance of Rubisco activation at high temperature suggests that Rca‐α has a functional thermo‐protective role in carbon fixation in C₄ grasses by sustaining Rubisco activation at high temperature.     

The temperature response of photosynthesis in tobacco with reduced amounts of Rubisco

The reasons for the decline in net CO₂ assimilation ( A ) above its thermal optimum are controversial. We tested the hypothesis that increasing the ratio of Rubisco activase to Rubisco catalytic site concentration would increase the activation state of Rubisco at high temperatures. We measured photosynthetic gas exchange, in vivo electron transport ( J ) and the activation state of Rubisco between 15 and 45 °C, at 38 and 76 Pa ambient CO₂, in wild-type (WT) and anti- rbc S tobacco. The Rubisco content of the anti- rbc S lines was 30% (S7-1) or 6% (S7-2) of WT, but activase levels were the same in the three genotypes. Anti- rbc S plants had lower A than WT at all temperatures, but had a similar thermal optimum for photosynthesis as WT at both CO₂ levels. In WT plants, Rubisco was fully activated at 32 °C, but the activation state declined to 64% at 42 °C. By contrast, the activation state of Rubisco was above 90% in the S7-1 line, between 15 and 42 °C. Both A and J declined about 20% from T _opt to the highest measurement temperatures in WT and the S7-1 line, but this was fully reversed after a 20 min recovery at 35 °C. At 76 Pa CO₂, predicted rates of RuBP regeneration-limited photosynthesis corresponded with measured A in WT tobacco at all temperatures, and in S7-1 tobacco above 40 °C. Our observations are consistent with the hypothesis that the high temperature decline in A in the WT is because of an RuBP regeneration limitation, rather than the capacity of Rubisco activase to maintain high Rubisco activation state.     

Rubisco activase is a key regulator of non-steady-state photosynthesis at any leaf temperature and, to a lesser extent, of steady-state photosynthesis at high temperature

The role of Rubisco activase in steady-state and non-steady-state photosynthesis was analyzed in wild-type (Oryza sativa) and transgenic rice that expressed different amounts of Rubisco activase. Below 25°C, the Rubisco activation state and steady-state photosynthesis were only affected when Rubisco activase was reduced by more than 70%. However, at 40°C, smaller reductions in Rubisco activase content were linked to a reduced Rubisco activation state and steady-state photosynthesis. As a result, overexpression of maize Rubisco activase in rice did not lead to an increase of the Rubisco activation state, nor to an increase in photosynthetic rate below 25°C, but had a small stimulatory effect at 40°C. On the other hand, the rate at which photosynthesis approached the steady state following an increase in light intensity was rapid in Rubisco activase-overexpressing plants, intermediate in the wild-type, and slowest in antisense plants at any leaf temperature. In Rubisco activase-overexpressing plants, Rubisco activation state at low light was maintained at higher levels than in the wild-type. Thus, rapid regulation by Rubisco activase following an increase in light intensity and/or maintenance of a high Rubisco activation state at low light would result in a rapid increase in Rubisco activation state and photosynthetic rate following an increase in light intensity. It is concluded that Rubisco activase plays an important role in the regulation of non-steady-state photosynthesis at any leaf temperature and, to a lesser extent, of steady-state photosynthesis at high temperature.     

Structure and functional annotation of hypothetical proteins having putative Rubisco activase function from Vitis vinifera

Rubisco is a very large, complex and one of the most abundant proteins in the world and comprises up to 50% of all soluble protein in plants. The activity of Rubisco, the enzyme that catalyzes CO₂ assimilation in photosynthesis, is regulated by Rubisco activase (Rca). In the present study, we searched for hypothetical protein of Vitis vinifera which has putative Rubisco activase function. The Arabidopsis and tobacco Rubisco activase protein sequences were used as seed sequences to search against Vitis vinifera in UniprotKB database. The selected hypothetical proteins of Vitis vinifera were subjected to sequence, structural and functional annotation. Subcellular localization predictions suggested it to be cytoplasmic protein. Homology modelling was used to define the three-dimensional (3D) structure of selected hypothetical proteins of Vitis vinifera. Template search revealed that all the hypothetical proteins share more than 80% sequence identity with structure of green-type Rubisco activase from tobacco, indicating proteins are evolutionary conserved. The homology modelling was generated using SWISS-MODEL. Several quality assessment and validation parameters computed indicated that homology models are reliable. Further, functional annotation through PFAM, CATH, SUPERFAMILY, CDART suggested that selected hypothetical proteins of Vitis vinifera contain ATPase family associated with various cellular activities (AAA) and belong to the AAA+ super family of ring-shaped P-loop containing nucleoside triphosphate hydrolases. This study will lead to research in the optimization of the functionality of Rubisco which has large implication in the improvement of plant productivity and resource use efficiency.     

The drelationship between CO2-assimilation rate,Rubisco carbamylation and Rubisco activase content in activase-deficient transgenic tobacco suggests a simple model of activase action

Transgenic tobacco (Nicotiana tabacum L. cv. W38) plants with an antisense gene directed against the mRNA of ribulose-1,5-bisphosphate carboxylase/ oxygenase (Rubisco) activase were used to examine the relationship between CO₂-assimilation rate, Rubisco carbamylation and activase content. Plants used were those members of the r₁ progeny of a primary transformant with two independent T-DNA inserts that could be grown without CO₂ supplementation. These plants had from < 1% to 20% of the activase content of control plants. Severe suppression of activase to amounts below 5% of those present in the controls was required before reductions in CO₂-assimilation rate and Rubisco carbamylation were observed, indicating that one activase tetramer is able to service as many as 200 Rubisco hexadecamers and maintain wild-type carbamylation levels in vivo. The reduction in CO₂-assimilation rate was correlated with the reduction in Rubisco carbamylation. The anti-activase plants had similar ribulose-1,5-bisphosphate pool sizes but reduced 3-phosphoglycerate pool sizes compared to those of control plants. Stomatal conductance was not affected by reduced activase content or CO₂-assimilation rate. A mathematical model of activase action is used to explain the observed hyperbolic dependence of Rubisco carbamylation on activase content.Abbreviations CA1P 2-carboxyarabinitol-1-phosphate - P_ip_a intercellular, ambient partial pressure of CO₂ - PGA 3-phospho-glycerate - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate - SSU small subunit of Rubisco     

Changes in protein quantities of phosphoenolpyruvate carboxylase and Rubisco activase in various wheat genotypes

In early seedlings of wheat genotypes two isoforms of Rubisco activase with molecular weights of 42 and 46 kDa are expressed. Amounts of both isoforms significantly increase in early seedlings of the durum wheat genotype Barakatli-95 exposed to salt stress. But at the beginning of the tillering stage, the changes in quantities of both RCA isoforms are different in durum and bread wheat genotypes subjected to a 3-day drought stress. In the leaves of the early seedlings of the studied wheat genotypes exposed to drought stress quantities of PEPC subunits increase compared to the control but they remain relatively stable in early roots and germinating seeds. However, quantities of its subunits decrease sharply in roots and germinating seeds of early seedlings under the influence of 100 mM NaCl. In flag leaves and ear elements of the Barakatli-95 genotype grown under normal water supply conditions protein quantities of PEPC subunits change differently depending on time. Changes in protein quantities of RCA, PEPC and Rubisco enzymes have been studied comparatively in ear elements and flag leaves after the fourth day of anthesis.     

An isoleucine residue acts as a thermal and regulatory switch in wheat Rubisco activase

The regulation of Rubisco, the gatekeeper of carbon fixation into the biosphere, by its molecular chaperone Rubisco activase (Rca) is essential for photosynthesis and plant growth. Using energy from ATP hydrolysis, Rca promotes the release of inhibitors and restores catalytic competence to Rubisco‐active sites. Rca is sensitive to moderate heat stress, however, and becomes progressively inhibited as the temperature increases above the optimum for photosynthesis. Here, we identify a single amino acid substitution (M159I) that fundamentally alters the thermal and regulatory properties of Rca in bread wheat (Triticum aestivum L.). Using site‐directed mutagenesis, we demonstrate that the M159I substitution extends the temperature optimum of the most abundant Rca isoform by 5°C in vitro, while maintaining the efficiency of Rubisco activation by Rca. The results suggest that this single amino acid substitution acts as a thermal and regulatory switch in wheat Rca that can be exploited to improve the climate resilience and efficiency of carbon assimilation of this cereal crop as temperatures become warmer and more volatile.     

Regulation of Rubisco activation in antisense plants of tobacco containing reduced levels of Rubisco activase

Following an increase in photon flux density (PFD), ribulose bisphosphate carboxylase/oxygenase (Rubisco) undergoes a slow activation which substantially limits the rate of photosynthesis. This activation process is mediated in part by Rubisco activase. Antisense DNA plants of tobacco were used to quantify the degree to which activase limits Rubisco activation. Reductions in leaf activase content caused proportional reductions in the rate of Rubisco activation following a PFD increase from 110 to 1200 micromol m(-2) sec(-1). This was the case for activase levels up to and slightly beyond normal wild-type activase levels. Activase therefore has a flux control coefficient of unity with respect to the Rubisco activation flux. Such a high control coefficient has rarely been measured for any metabolic system, and this is the highest control coefficient measured for an important photosynthetic flux. In contrast, the rate of Rubisco inactivation in leaves following a drop in PFD of 1200 to 110 micromol m(-2) sec(-1) was unchanged by a 60% reduction in activase levels. Despite the high degree of control that activase exerts over the rate of activation, and thus non-steady-state photosynthesis, it was shown that steady-state photosynthesis was largely unaffected by activase concentration until it was reduced below approximately 15% of the wild-type level. The significance of these results and their implications for published models of Rubisco activation are discussed.     

Influence of exogenous application of glutathione on rubisco and rubisco activase in heavy metal-stressed tobacco plant grown in vitro

The effect of glutathione on the influences of heavy metals affecting rubisco and rubisco activase was studied in tobacco plants grown in vitro where the shoot explants of the tobacco plant cultured on MS medium under aseptic conditions and two explants were placed in the control, 0.1 mM GSH, 1 mM GSH, 0.2 mM Cd, 0.2 mM Cu, 0.2 mM Zn, and a mixture of Cd and GSH, Cu and GSH, Zn and GSH, respectively. The effect of GSH on the growth of the tobacco plant was minimal, but the heavy metals clearly retarded its growth. GSH recovered the growth retarded by heavy metals, and the concentration of GSH required to recover the growth differed depending on the heavy metals. The content of chlorophyll in the plant increased through GSH and Zn, and decreased through Cd and Cu. The chlorophyll content which decreased due to Cd and Cu was recovered by GSH, and the content which increased due to Zn was decreased by 1 mM GSH. The content of rubisco decreased due to GSH and heavy metals, and the content which decreased due to heavy metals was recovered by GSH, and when GSH was treated with Zn, the increased rate was maximum compared to other heavy metals. The activity of rubisco was increased due to GSH and heavy metals, and the activity increased by Cd and Zn decreased through GSH. In the case of Cu, the activity of GSH increased even more. There was no effect of GSH on the influences of heavy metals on the content and activity of rubisco activase. The activity of rubisco decreased by thiourea among six denaturing agents, and increased by l-cysteine, and in most cases the activity level was recorded as high. The activity of rubisco activase all decreased as a result of six denaturing agents, and the effect caused by EDTA and guanidine-HCl was the greatest, while the effect caused by l-cysteine and urea was minimal.     

Detection of Rubisco and mycotoxins as potential contaminants of a plantibody against the hepatitis B surface antigen purified from tobacco

Antibodies have been one of the proteins widely expressed in tobacco plants for pharmaceutical purposes, which demand contaminant free preparations. Rubisco constitutes 40-60% of tobacco leaf soluble proteins; therefore it is the major potential protein contaminant of plantibodies, while mycotoxins are toxic compounds that could be introduced during the biomass production and post-harvest stages with important consequences to human health. The objective of this paper was to investigate whether Rubisco and mycotoxins are present in Plantibody HB-01 preparations used in the immunopurification of the hepatitis B surface antigen. Rubisco was purified from Nicotiana tabacum yielding 154 microg of protein per gram of leaves and purity over 95%. Among mouse monoclonal antibodies generated against this enzyme, the CBSS.Rub-2 was selected for its immunodetection. It recognizes a conserved sequential epitope of Rubisco large subunit with an affinity constant of 0.13 x 10(8)M(-1). Rubisco quantification limit was 1 microg spreading to the measurement of this contaminant less than 4% of plantibodies samples. Additionally, according to a Reverse Phase-HPLC used to measure the level of adventitiously introduced contaminants, it can be concluded that aflatoxins B1, B2, G1 and G2 were undetected in the purified Plantibody HB-01 samples.     

Two conserved tryptophan residues are responsible for intrinsic fluorescence enhancement in Rubisco activase upon ATP binding

Two species-invariant tryptophan residues at positions 109 and 250 of tobacco Rubisco activase were identified by site-directed mutagenesis as being responsible for the increase in intrinsic fluorescence upon addition of ATP, which has been previously attributed to increased self-association. Substitution of W109, which is immediately prior to a ‘P-loop’ sequence in the ATP catalytic motif, with aromatic residues (Tyr or Phe), Cys or Lys eliminated both ATP hydrolysis and the intrinsic fluorescence enhancement. Although the W109 mutants bound ATP, ATP did not provide a partial protection against proteolysis by trypsin that was observed with the recombinant wild-type enzyme. In contrast, substitution of W250 with Tyr or Phe abolished about half (44%) of the increase in intrinsic fluorescence with ATP, but had little effect on ATP hydrolysis, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activation or proteolytic protection with ATP. The substitution of the other tryptophan residues, W16 and W305, with phenylalanine did not significantly alter the change in intrinsic fluorescence upon addition of ATP. Therefore, W109 and W250 are the residues reporting the conformational change that increases the intrinsic fluorescence.     

Influence of salicylic acid on rubisco and rubisco activase in tobacco plant grown under sodium chloride in vitro

The present study was designed to evaluate the influence of salicylic acid (SA) on the growth of salt stress (sodium chloride) induced in tobacco plants. In addition, quantification of rubisco and rubisco activase contents of the plants was also determined in treatments with the control, 10⁻⁴ mM SA, 50 mM NaCl, 100 mM NaCl, 150 mM NaCl, SA + 50 mM NaCl, SA + 100 mM NaCl and SA + 150 mM NaCl, respectively after in vitro culture for 5 weeks. The growth of the tobacco plant decreased in 50 mM and 100 mM NaCl when not treated with SA. However, the growth was accelerated by SA, and the growth retardation caused by NaCl was improved by SA. The content of rubisco was improved by SA only in plants treated with 50 mM NaCl, and the activity of rubisco was increased by SA resulting in the decreased effect of NaCl, but only in 50 mM NaCl treated plants. The content of rubisco activase decreased due to NaCl, and SA did not improve the effect caused by NaCl. The activity of rubisco activase was increased by SA resulting in decreased activity caused by NaCl, but increased effect by SA was not recovered to the level of NaCl untreated plants. The activity of rubisco and rubisco activase, which decreased due to denaturing agents, did not demonstrate significant improvement when compared to the control.     

Increased zinc content in transplastomic tobacco plants expressing a polyhistidine-tagged Rubisco large subunit

Rubisco is a hexadecameric enzyme composed of two subunits: a small subunit (SSU) encoded by a nuclear gene (rbcS), and a large subunit (LSU) encoded by a plastid gene (rbcL). Due to its high abundance, Rubisco represents an interesting target to express peptides or small proteins as fusion products at high levels. In an attempt to modify the plant metal content, a polyhistidine sequence was fused to Rubisco, the most abundant protein of plants. Plastid transformation was used to express a polyhistidine (6x) fused to the C-terminal extremity of the tobacco LSU. Transplastomic tobacco plants were generated by cotransformation of polyethylene glycol-treated protoplasts using two vectors: one containing the 16SrDNA marker gene, conferring spectinomycin resistance, and the other the polyhistidine-tagged rbcL gene. Homoplasmic plants containing L8-(His)6S8 as a single enzyme species were obtained. These plants contained normal Rubisco amounts and activity and displayed normal photosynthetic properties and growth. Interestingly, transplastomic plants accumulated higher zinc amounts than the wild-type when grown on zinc-enriched media. The highest zinc increase observed exceeded the estimated chelating ability of the polyhistidine sequence, indicating a perturbation in intracellular zinc homeostasis. We discuss the possibility of using Rubisco to express foreign peptides as fusion products and to confer new properties to higher plants.